For the purposes of the present invention “glucuronic acid” and “4-O-methyl glucuronic acid” are referred to hereinbelow by the terms “GlcA” and “MeGlcA”, respectively. The term “[Me]GlcA” is used as a collective noun herein to denote both GlcA and MeGlcA.
Native xylan is a hemicellulose that is often cross-linked to lignin via [Me]GlcA side chains (e.g. Balakshin et al. 2007, Holzforschung, 61, 1-7). Native xylan is also tightly associated with cellulose microfibrils. Native xylan is of value in certain industries but its extraction is complicated by a range of covalent and non-covalent chemical linkages to other components of the cell wall, such as linkages to [Me]GlcA. Typically, enzymatic and chemical means are used inter alia, to disrupt the side chain linkages, enabling the extraction of xylan. Extraction processes are generally costly and give rise to side products that are undesirable.
Enzymatic methods are also used to depolymerise the cellulose and hemicelluloses into soluble hexose and pentose sugars. Depolymerisation (also referred to in the art as “saccharification”) of xylan requires a multiplicity of enzymes to break the backbone and side chain linkages. Some of the products of enzymatic treatments are not able to be used by many organisms used in fermentation or in bio-processing to produce liquid transport fuels such as ethanol or butanol.
Extracted native xylan is used inter alia in paper production and modified xylan-containing plant material has potential for use in, inter alia, the production of sugars and indirectly, in the production of liquid transport fuels such as ethanol via fermentation of the sugars. Cellulose fibrils, used inter alia in paper and other materials, are damaged by the processes of extraction of xylan and other hemicellulose from the fibrils.
The prior art appears to be silent about the precise role that xylan saccharide side chain modifying enzymes, such as XGAT enzymes, play in secondary cell wall structure, and their function does not appear to have been accurately elucidated.
Brown D. M. et al The Plant Cell, Vol. 17, 2281-2295, August 2005 describes a study relating to mutant Arabidopsis thaliana plants comprising insertions into a so-called glycosyl transferase 8-like gene, identified as At3g18660, which allegedly resulted in a plant that had a weak stem, a feature known to be a characteristic of known secondary cell wall mutants. However, the elucidation of the function of At3g18660 and the role it plays in secondary cell wall synthesis does not appear to be described by Brown D. M. et al. Indeed, At3g18860 is not described as a xylan glucuronyl transferase and no putative industrial use for At3g18660 is contemplated.
Pena M. J. et al The Plant Cell, Vol. 19:549-563, February 2007 describe studies on mutant plants of Arabidopsis thaliana in which the presence of “wood-associated GTs”, such as IRX8 are present. The study is confined to looking at IRX8 and IRX9 genes and their effect on glucuronoxylan chemistry and structure. Arabidopsis thaliana genes At4g33330 and At3g18660 are mentioned as homologs (sic) of “wood associated GTs” but that appears to be all that Pena M. J. et al supra say about them.
Zhong R. et al The Plant Cell Vol. 17, 3390-3408, December 2005 describe studies on inter alia mutant plants of Arabidopsis thaliana Fragile Fiber8 which is thought by the authors to encode a glucuronyl transferase that is involved in secondary wall synthesis. Zhong R et al report that the GlcA component of xylan is missing. It is not reported that the level of overall [Me]GlcA substitution is unchanged. The data presented by Zhong R et al indicates that they were unable to distinguish if the level of overall [Me]GlcA substitution had changed. In wild type plants, the xylose in the xylan is about 7% substituted with MeGlcA and 3% with GlcA; 10% overall. Thus, the overall proportion of xylose substitution is no different to wild type plants. The overall substitution pattern is unlike the new xylan structures created using the XGATs of the invention in which the absolute substitution level of [Me]GlcA on the xylans created using At4g33330 and/or At3g18660 can be varied depending on the level of expression or the level of gene silencing that may be generated.